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POTENTIAL FOR REDUCING SURVIVAL OF SCLEROTIA OF SCLEROTIUM
CEPIVORUM IN THE FRASER VALLEY OF BRITISH COLUMBIA
VJPPEN K. JOSHI
B.Sc. Medical Sciences, 1981, Guru Nanak Dev University, India
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF '
MASTER OF SCIENCE
in the Department
of
Biological Sciences
@ VIPPEN K. JOSHI 1988
SIMON FRASER UNIVERSITY
August 1988
All rights reserved. This work may not be reproduced in whole or in part by photocopy
or other means, without permission of the author.
APPROVAL
Name:
Degree :
Vippen Kumari Joshi
Master of Science
Title of Thesis:
POTENTIAL FOR REDUCING SURVIVAL OF SQ;EROTIA OF SQ;EROTIUM-CEPIVORUM IN THE FRASER VALLEY OF B. C.
Examining Comnittee:
Chairman : Dr. R.C. Brooke, Associate Professor
DL J.E. Rahe, Professor, Senior Supervisor
Dr. G'.R. ~ister, ASS^?^^^ Professor
-~ Dr- J. Menzies, ~eseardh Scientist, Plant Pathology, Agriculture Canada, P.O. Box 1000, Agassiz, B.C., VOM IAO, Public Examiner
PARTIAL COPYRIGHT LICENSE
I horoby g ran t t o S lmon. Frasor Un l v a r s 1 t y tho r I gh t t o i end
my thesis, proJoct o r oxtandod ossay ' ( th0 : I t l o o f which Is shown bolow)
t o users o f tho Simon Frasor Un l vo rs l t y ~ l b r i r ~ , and t o m k o p a r t i a l o r
s ing le coples on ly f o r such usors o r I n rasponsa t o a requast from tho
l i brary o f any o thor un lvo rs l t y , or o thor oducat lona 1 I n s t l t u t Ion, on
i t s own bohalf o r f o r ono o f I t s usors. I f u r t h o r agreo t h a t porrnission
f o r m u l t l p l o copying o f t h l s work f o r scho la r l y purposos may bo grantod
by mo o r tho Doan o f Graduato Studios. I t I s -understood t h a t . copy1 ng
o r pub1 l c a t l o n o f t h l s work f o r f lnanc la l gain, shol 1 not bo a l iowod - wl thout my w r l t t e n permlsslon.
b
C.
T l t l e of Thas I s/ProJect/Extandod Essay
Potential for reducing survival of sclerotia of Sclerotium-cepivorum
in the Fraser Valley of B.C.
Author:
( i l g n a t u r o )
V i ~ ~ e n Kumari Joshi
ABSTRACT
The potential of using agronomic practices for reducing the survival of sclerotia of
Sclerotium cepivorum in the field and hence protecting onions from white rot, a disease
caused by this pathogen was investigated.
Indigenous sclerotial populations of S. cepivorum were estimated in a commercial
onion field near Cloverdale, B.C. after harvest of a crop which had suffered 22-30%
losses to white rot in 1986. The effect of the use of Brassica /itncea as a winter cover
crop and its residue incorporation on the su~vival of sclerotia of S. cepivorum in this
field was studied. Survival of indigenous and introduced sclerotial populations was assessed
at different times of the year. There was a significant decline in number of sclerotia with
time in all plots. A significantly greater decline in the numbers of intact firm sclerotia
occurred in B. /itncea treatment plots than in control plots.
The level of disease in a commercial onion crop grown on the study site the
following season was assessed. The results obtained showed that despite the substantial
decrease in inoculum levels of sderotia in treated plots, the diseasc incidence was not C
significantly different in treated and control plots.
In another experiment the effect of flooding on survival of sclerotia of S. cepivorum
in non-amended soil and soil amended with B. /itncea or natural vegetation was assessed.
Flooding reduced sclerotial survival but the magnitude of this effect was related to the
presence and nature of the vegetative amendments. The results obtained lead to a
conclusion that a combination of flooding and specific crop residue incorporation can
reduce survival of sclerotia in the soil under the type of conditions (mild winter with
high precipitation) experienced in the Fraser Valley of B.C. The study did not detect a
reduction in white rot of onions associated--with reduced survival of sclerotia. The
iii
implications of the utilization of flooding and crop residue incorporation in reducing
sclerotial survival are discussed in relation to other disease management methods.
ACKNOWLEDGEMENTS
I would like to express my deepest gratitude and appreciation to Dr. James E.
Rahe, my Senior Supervisor for his very wise guidance, encouragement, patience and
continuous help at each ind every stage of this project Much thanks to Dr. G.R. Lister
for his valuable suggestions and encouragement
Special thanks to Mr. H.D. Pierce, Jr. for his technical help. I wish to thank my
friends Eric Littley and Andre Levesque for their thoughtful suggestions and their advice
on statistical analysis; Omer Medani for his help in setting up field experiment; Mr.
Andrew Stetar for providing space on his farm for field studies and Sandra Zervos for
her moral support.
I wish to express my profound gratitude to my sister Adarsh Sharma and her
husband Dharmpal Sharma who sponsored me to come to Canada and all my brothers
and sisters from Canada and abroad who inspired, encouraged and helped me to get
higher education since my childhood. A very special tribute to my father Mr. Bhagat Ram C
Joshi and my mother Mrs. Sarla Joshi for their enormous help in every sphere of my A
life and without whose encouragement -1 could not have accomplished this work.
DEDICATION
TO MY PARENTS
TABLE OF CONTENTS
Approval ...................................................................................................................................................................... ii
ABSTRACT ............................................................................................................................................................... iii
ACKNOWLEDGEMENTS .. .................................................................................................................................... DEDICATION ..........................................................................................................................................................
......................................................................................................................................................... List of Tables
List of Figures .........................................................................................................................................................
1 . DISEASE HISTORY AND INTRODUCTION TO THE THESIS .......................................
1.1 WHITE ROT ...............................................................................................................................
1.2 PATHOGEN .................................................................................................................................
1.3 SURVIVAL OF THE PATHOGEN ..................................................................................
1.4 INFECTION AND DISEASE DEVELOPMENT ..........................................................
1.5 SYMPTOMS OF THE DISEASE ......................................................................................
1.6 CHEMICAL CONTROL ................... : ......................................................................................
1.7 FACTORS AFFECTING GERMINATION OF SCLEROTIA ................................
1.8 .I BIOLOGICAL CONTROL ...................................................................................................
2 . EFFECT OF BRASSICA JUNCEA AS A WINTER COVER CROP ON . THE SURVIVAL OF SCLEROTIUM CEPIVORUM ...........................................................................
2.1 INTRODUCTION ....................................................................................................................
2.2 MATERIALS AND METHODS .......................................................................................
2.3 RESULTS ....................................................................................................................................
2.4 DISCUSSION .............................................................................................................................
3 . SURVEY OF DISEASE LEVEL IN COMMERCIAL ONION FIELD ..........................
3.1 INTRODUCTION ....................................................................................................................
3.2 METHODS FOR ESTIMATING DISEASE LEVEL ...............................................
3.3 RESULTS ....................................................................................................................................
vii
3.4 DISCUSSION ............................................................................................................................. 38
4 . EFFECT OF FLOODING AND ORGANIC AMENDMENTS ON SURVIVAL OF SCLEROTIA OF SCLEROTIUM CEPIVORUM ...............................................................
4.1 INTRODUCTION ....................................................................................................................
4.2 MATERIALS AND METHODS .......................................................................................
4.3 RESULTS ....................................................................................................................................
4.4 DISCUSSION .............................................................................................................................
5 . GENERAL DISCUSSION AND CONCLUSIONS .....................................................................
BIBLIOGRAPHY ....................................................................................................................................................
Index ...........................................................................................................................................................................
viii
Table
LIST OF TABLES
Page
Indigenous populations of sclerotia of Sclerotium cepivwum estimated at 0, 171 and 272 days after seeding with Brassica /itncea ................................................. 22
............... . Temperature and rainfall data for the period September, 86 to June, 87. 23
Comparison of proportions of sites with white rot at different sampling times in Brassica treated and control subplots. ..................................................................... 34
Comparison of percentage of plants with white rot at different sampling times in Brassica treated and control plots. ........................................................................... 35
Comparison of percent smut at different times in Brassica treated and control plots. ............................................................................................................................................ 36
Comparison of disease levels and yield of onions at harvest in Brassica treated .................................................................................................................... and control plots. 37
Figure
LIST OF FIGURES
Page
An onion bulb showing symptoms of white rot ........................................................................ 5
Chemical structure of dliin, a component of root exudates of Allium spp. ............... 7
Hypothetical scheme showing three sources of volatile organic compounds produced in soil (A,B,C) and their possible effect and modes of action on propagules of soil-borne fungal pathogens in soil. ........................................... 9
Field experiment showing arrangement of nylon mesh bags containing sclerotia of Sclerotium cepivorum in a study plot ................................................................. 19
Effects of Brassica Jitncea, natural conditions and rototilling on persistence of indigenous populations of sclerotia of Sclerotium cepivorum in Fraser Valley muck soil. .................................................................................................................. 24
Effects of Brassica Jitncea and natural conditions on the survival of introduced sclerotia of Sclerotium cepivorum in Fraser Valley muck soil. ....................... 25
Dual culture test on potato dextrose agar showing antagonism between Sclerdium cepivorum and an unidentified bacterium isolated from the surface of sclerotia of S. cepivwum. .......................................................................... 26
Styrofoam cups to show the cut and intact bottom used for drained and flooded condition, respectively. ........................................................................................... 44
Diagramtic representation of arrangement of nylon mesh bags containing sclerotia of Sclerotium cepivorum and p l a t material in the soil in
C cups. ............................................................................................................................................. 45 *
Experimental setup showing flooded & nonflooded treatments in a randomized '
complete block design. .......................................................................................................... 47
Effects of organic amendments on survival of introduced populations of sclerotia of S. cepivorum under the influence of flooded or nonflooded soil. ......... 49
CHAPTER 1
DISEASE HISTORY AND INTRODUCTION TO THE THESIS
1.1 WHITE ROT
White rot is a serious disease of onion and other Allium spp. caused by the soil
borne fungus, Sclerdium cepivwum Berk. (Coley-Smith 1959; Walker 1969; Jones and
Mann 1963). It was first discovered in England by M.J. Berkeley in 1841 and has since
been reported in most commercial onion production areas in many countries (Walker 1969;
Scott 1956; Adam & Papavizas 1971). It can cause severe yield losses (Adams 1981;
Crowe et d. 1980; Croxall et d. 1953).
Dry bulb and green bunching onions are grown in the Fraser Valley of British
Columbia. Spring seeded dry bulb onions are one of the leading vegetable crops grown
on muck soils near Cloverdale, British Columbia (Rutherford & Ormrod 1985). White rot
was first recorded in the commercial onion growing areas of the Fraser Valley in 1970
(Ormrod & Conroy 1970). By 1974 the disease had spread to every pnion field on the
farm where it was first recorded resulting in a loss of 50% of the crop (Orrnrod et al. '
1977). In the spring of 1975, regulations were passed under the British Columbia Plant
Protection Act forbidding the growing of onions on land known to contain S. cepivorum
and restricting the movement of soil and machinery from such lands (Onnrod et al.
1977). However the disease has continued to spread and is a major problem to the local
coinmercial onion industry (Utkhede et al. 1978).
White rot of onions is a major problem for the growers because it continues to
spread and increase in severity where onions are grown in infested soil. It causes
substantial yield losses and no completely effective chemical or biological control is yet
available (Utkhede 1982).
1.2 PATHOGEN
S. cepivorum is a facultative saprophytic fungus (class Deuteromycetes; order Mycelia
Sterilia) and grows in soil from an infected food base (Scott 1956). Its pathogenic
- activities under natural conditions are restricted to Allium spp. (Coley-Smith 1959, 1979).
The fungus produces microconidia in nature (Coley-Smith 1960) and in culture (Asthma
1947) but these spores have not been observed to germinate and their function is
unknown. In the absence of the host, the pathogen usually persists in soil as a sclerotium
for long periods without any known vegetative growth or functional sportdating stage
(Coley-Smith 1959; Walker 1969; Crowe et al. 1980).
1.3 SURVIVAL QJ PATHOGEN
The only means by which the pathogen is known to survive in the absence of a
host is as small black (0.2 to 0.6 mm diam.) sclerotia (Coley-Smith 1959; Crowe et al. -
1980; Walker 1969). These propagules are extremely persistent and remain viable in soil
for many years even in the absence of host Allium spp., either on or beneath the soil A
surface (Coley-Smith 1959; Walker 1969; Crowe et al. 1980). Coley-Smith (1979) recovered
sclerotia from soil even after 10 years burial, and Crowe et al. (1980) recovered viable
sclerotia from fields in California where no onions had been grown for 15 years. In
contrast Leggett et al. (1983) reported a substantial decay of sclerotia within one year of
burial in muck soil in the Fraser Valley of B.C. The rapid decay of sclerotia was
presumably due to the local environmental conditions.
The sclerotium has an outer rind of one or two layers of rounded, thick cells
enclosing a large tissue of hyphae. They are formed in diseased tissue on the onion stem
base and are released into the soil either when the infected plant decays or at harvest
time (Coley-Smith 1959; Entwistle & Munasinghe 1978). Sclerotia can withstand the
extremes of wetness and dryness in the soil (Coley-Smith et d. 1974; Papavizas 1977).
The sclerotia remain dormant in nonsterile soil until stimulated to germinate by root
exudates from Allium spp. (Coley-Smith et d. 1968; Coley-Smith & Hickrnan 1957;
Coley-Smith & King 1969; 'Elnaghy et d. 1971; King & Coley-Smith 1969a).
1.4 INFECTION DISEASE DEVELOPMENT
Primary infection of the host plant is by hyphae originating from germinating
sclerotia of S. cepivorum (Coley-Smith 1959; Walker 1969; Crowe et d. 1980). Sclerotia
germinate within a temperature range of 14• ‹~-200C (Adams & Papavizas 1971; Entwistle
& Munasinghe 1978). Two types of germination have been reported: hyphal germination
(Adams & Papavizas 1971; Coley-Smith et d. 1967), and eruptive or plug germination
(Coley-Smith 1960). The eruptive germination is generally more infective than hyphal
germination (Scott 1956; Coley-Smith 1960).
Roots of onion bulbs are attacked by the fungus, which subsequptly invades and
forms a superficial white, fluffy mycelium on the lower portion of the bulb. After the A
primary infection, the mycelial growth can spread from one plant to another, thus causing
secondary infections (Crowe & Hall 1980a; Scott 1956). Hyphae of S. cepivorum spread
intercellularly and intracellularly through roots and cortical tissue of stems, eventually
destroying the parenchymatous tissue (Abd-El-Razik et d. 1973). New sclerotia are formed
within and on the host tissue and become distributed across the fields and through the
soil profile in subsequent years (Crowe et d. 1980).
1.5 SYMPTOMS DISEASE
The above ground symptoms of white rot are yellowing and dying of the outer
leaves of the onion plant beginning at the tips and progressing downwards (Walker 1969).
The disease may occur early in the season and cause death of seedlings, but significant
losses are more commonly observed in late summer as a bulb rot The root system is
rapidly destroyed by the fungus and infected bulbs become soft and watersoaked in the
area colonized by the pathogen. Example of an infected bulb is shown in Fig. 1.
1.6 CHEMICAL CONTROL
Many control measures for white rot have been tried in different parts of the
world, with limited success. In some cases where infestation of the soil is severe, onion
production has had to be abandoned (Adarns and Papavizas 1971; Merriman et al. 1980).
Neither chemical or biological treatment has reduced the disease to economically acceptable
levels in the Fraser Valley of B.C.
Control of white rot by chemicals, including calomel (Booer, 1945; Croxall et al.
1953), dichloran (Fletcher et al. 1971), pentachloronitrobenzene (PCNB) (Rushdi et d.
1974), iprodione (Entwistle & Munasinghe 1980; Utkhede & Rahe 1979) and vinclozolin
(Utkhede & Rahe 1979) has been reported but none of these is considered to provide
reliable economic control. Among these chemicals, only dichloran is registered for use on
onions in Canada. The dicarboximide fungicides iprodione and vinclozolin have significantly
reduced white rot in field trials in the Fraser Valley (Utkhede & Rahe 1979) and might
provide a short term solution to the white rot problem. Dicarboximides likely could not
be relied upon in the long term because there is already evidence that the pathogen can
develop resistance to them (Littley & Rahe 1984), and loss of efficacy of iprodione due
Figure 1: An onion bulb showing symptoms of white rot
to enhanced degradation in soil was reported by Walker et al. (1986).
In view of these problems, other control methods such as breeding for resistance
and biological control are being sought Work done at Simon Fraser University has
identified sources of resistance to white rot (Utkhede & Rahe 1978). It would take several
years before a commercially acceptable cultivar could be developed, however, and it is
unlikely that the level of resistance that can be achieved will provide complete control.
There is thus need for increased effort to achieve the control of white rot of onions by
biological means.
1.7 FACTORS AFFECTING GERMINATION SCLEROTIA
The primary functions of the sclerotium are to perrenate the fungus, and to bring
about successful contact between the pathogen and host tissue. Therefore, sclerotial
germination is an important phase at which the control of S. cepivorum might be
implemented. There are three groups of factors which are known to influence the
germination of sclerotia of S. cepivorum.
1. Host plant exudates
2. Soil microflora as biotic soil factors
3. Abiotic soil factors.
Coley-Smith (1979) demonstrated that the dormancy of sclerotia of S. cepivmm in
the field is a condition imposed by the soil microflora, since they germinate readily in
sterile soil. They are held in a dormant condition by soil fungistasis (Allen & Young
1968; Coley-Smith et al. 1967; King & Coley-Smith 1969b) and are stimulated to
germinate by compounds associated with aqueous extracts and exudates of Allium spp.
(Coley-Smith 1960; Coley-Smith & Hickman 1957; Coley-Smith & Holt 1966). The active
volatile compounds in extracts of Allium spp. appear to be alkylsulphides and in particular
COOH
ALL YLCYSTEINE SULPHOXIDE I
(ALLI IN)
FIGURE 2: Chemical structure of alliin, a component of root exudates of Allium spp.
(King and Coley-Smith, 1969a)
n-propyl and allylsulphides (Coley-Smith & Cooke 1971). Sclerotia show a low response to
methyl sulphides which are produced by a wide variety of plants but a high response to
n-propyl and allylsulphides produced only by Allium spp. These volatile compounds are not
produced by intact onion plants. Intact onion plants exude small quantities of ally1 cysteine
sulphoxides. These latter (Fig. 2) compounds are thermostable, water soluble and diffusible,
and are metabolized by bacteria to generate stimulatory volatile alkylsulphides (King &
Coley-Smith 1969a).
The use of onion oil to induce germination of sclerotia in the absence of host
plants and thereby reduce the number of sclerotia has been evaluated (Merriman et d.
1980; Utkhede & Rahe 1982). Partial control of the disease was achieved presumably
because a substantial proportion of sclerotia germinated in the absence of the host,
decayed quickly and did not form secondary sclerotia. However control was incomplete and
implementation of this method would be prohibitively expensive unless a ' germination
stimulant much cheaper than onion oil could be produced.
An onion flavour component, namely diallyl-disulphide (DADS) has also been
evaluated for control of white rot under laboratory and field conditions (Entwistle et d. *
1982; Coley-Smith & Parfitt 1986). Variable efficacy was obtained and it was. found that
there was a marked seasonal response of S. cepivwum to treatment with DADS
(Coley-Smith & Parfitt 1986). There are a number of features of DADS treatment which
have to be resolved before it could be recommended to farmers for control of white rot
Volatile stimulants for sclerotial germination are produced from their precursors in
onion root exudates by bacterial degradation, thus soil microflora may play an important
role in the induction of sclerotial germination (Coley-Smith and Dickinson 1971; Dickinson
and Coley-Smith 1970; Reddy 1986) It has also been reported that sclerotia of S.
cepivorum enhance microbial activity (Coley-Smith & Dickinson 1971; Dickinson &
Coley-Smith 1970). It seems that soil microflora and/or microflora associated with sclerotia
Figure 3: Hypothetical scheme showing three sources of volatile organic compounds produced in soil (A,B,C) and their possible effect and modes of action on propagules of
soil-borne fungal pathogens in soil.
NON HOST ?*@PLANT RESIDUE
HOST ROOTS
I
DECOMPOSITION
VOLATILE AND NON VOLATILE
PRODUCTS
M ICROORGANISMS
PRODUCTS
VOLATILE / STIMULANTS
-ABC-
MYCOSPHERE SOIL
I - - * \ _ - -
POSSIBLE STIMULATORS OR INHIBITORS
SCLEROTIAL EXUDATES
N SCLEROTIUM
may mediate the stimulatory effect of Allium root exudates.
The possible sources and interactions of volatile stimulants, sclerotia and the soil
microflora are summarized in Fig. 3.
1.8 BIOLOGICAL CONTROL
Biological control by protection of host plant surfaces involves the establishment of
antagonists in or near the infection court of the host, thereby providing a biological
barrier against the target pathogen (Cook 1981~). Biological control of this type may
involve the use of antagonists as biological pesticides, some of which are commercially
available (De Trogolf & R i w d 1976; Greigg 1975; Ken 1980; Rishbeth 1963). Several
other diseases have been controlled biologically in field trials but these biological agents
have not yet been developed for commercial use (Backman & Rodrigues-Kabana 1975;
Harman et al. 1980; Kommendahl & Chang 1975).
Several soil microorganisms have been found to be antagonistic to mycelial growth of
S. cepivorum. Some Fusarium species, Penicillium nigricans (Moubashef et al. 1970) and P
Streptomyces griseus (Memman & Borkenhead 1977) have been used to .control S.
cepivorum with varying success. Other soil fungi such as Trichoderma viridae and
Coniothyrium minitans (Coley-Smith & Cooke 1971) are parasitic to sclerotia of S.
cepivorum and have been effective in control of S. cepivorum (Ahmed & Tribe 1977;
Abd-El-Moity et al. 1982).
The microorganisms that show substantial antagonistic activity against S. cepivorum in
laboratory or greenhouse experiments, few that have been tested in the field have given
disappointing results. Field trials in the Fraser Valley showed that Bacillus subtilis could
reduce the levels of white rot significantly in some cases (Utkhede & Rahe 1980) but
the results obtained from 4 years experimentation were not consistent A preliminary study
of the effect of vesicular arbuscular myconhizal (VAM) fungi on white rot did not reveal
potential for disease control (Tardif 1987), but much remains to be studied about the
possible interaction between VAM and onion white rot.
Abiotic soil factors such as temperature, moisture, pH and soil structure are other
factors which may affect either directly or indirectly the germination process. Changes in
these abiotic variables may affect both the activity of soil microflora and the host plant
exudation, thus influencing indirectly the germination of sclerotia.
The microbial activity of soil bacteria has been shown to increase when dry sclerotia
sclerotia of S. cepivorum are placed in unsterile soil (Coley-Smith & Dickinson 1971;
Dickinson & Coley-Smith 1970). Smith (1972a. 1972b. 1972c) claimed that air-dried sclerotia
of a number of fungi including S. cepivorum and S. rdfiii rot more quickly than do
non air dried sclerotia when placed in moist soil. He attributed the enhanced decay to
the intensified microbial activity caused by nutrient leakage from the dried sclerotia. In
contrast to Smith's results, Coley-Smith (1959) found that when dried sclerotia of S.
cepivorum were buried in field soil, a large proportion survived for 4 years.
C
Crowe & Hall (1980b) reported that wet and warm soil conditions promoted decay A
of sclerotia. Their results supported those of Scott (1956) who indicated that flooding
might eradicate sclerotia from infested soil. Leggett (1983) suggested that the unusually high
rate of decay of sclerotia within one year may be due to winter flooding usually
encountered in Fraser Valley muck soils, althogh no direct evidence was presented. Adarns
(1987) found that high soil temperatures (40-50~C) and low soil moisture reduced survival
and activity of sclerotia of S. cepivorum and S. minor.
There is less known about the optimum soil conditions for germination of sclerotia
of S. cepivorum than might seem to be the case because most of the optimum
conditions have been determined for disease development, rather than for sclerotial
germination (Entwistle & Munasinghe 1976; Walker 1926). Walker (1926) studied the
relation of soil temperature and soil moisture to onion white rot disease development. The
optimum temperature was found to be 20-24OC. Adam & Papavizas (1971) found that
the optimum temperatiwe for onion white rot development was 15OC, whereas for
germination of sclerotia of. S. cepivorum in autoclaved soil it was 20•‹C. Optimum pH for
growth of mycelium on agar has been found to be 4.8 to 5.3 but in the soil more
than 50% of the sclerotia germinated at pH between 4.5 and 7.8. Entwistle & Munasinghe
(1976) demonstrated that sclerotial germination, mycelial growth and seedling infection were
all markedly influenced by temperature. Crowe & Hall (1980b) reported that germination
varied with matric potential rather than with moisture content of two different soil types.
They found that decay of sclerotia of S. cepivorum was greatest at soil saturation but
this occurred only at high temperature. Sclerotial survival would appear to be affected by
the interaction of temperature and moisture levels.
As sclerotia are able to survive for many years in the absence of a host
(Coley-Smith 1959; Walker 1969; Crowe et al. 1980), crop rotation does not seem to
have much potential for reducing populations of sclerotia. Some cultural practices such as @
the use of organic amendments, altering sowing times (Rushdi et al. 1974) and the use ,
of onions as a trap crop for reducing the numbers of sclerotia of S. cepivohm in soil
have been tried but have been of very little success in controlling white rot (Merriman
& Issacs 1978).
Coley-Smith and Parfitt (1983) reported that allylisothiocyanate (ACN) was quite
effective in stimulating sclerotial germination. Surprisingly this chemical is not found in
Allium spp., but does occur in certain species of the family Cruciferae (Itoh et al. 1984,
1985; Kozima et al. 1987; Lewis & Papavizas 1971; Lin & Hua 1986) and specifically in
Brassica juncea (mustard, Wallbank and Wheatly 1976). Studies have shown that during the
decomp0siti6~ of crucifers in soil, some volatile compounds with phytotoxic as well as
mycotoxic properties are produced (Lewis & Papavizas 1971; Patrick et al. 1964). It is
possible that certain crucifers may be able to influence the behaviour or survival of S.
cepivorum under field conditions.
The purpose of this thesis was to examine the effects of Brassica /itncea and
flooding on the survival of indigenous and artificially introduced populations of sclerotia of
Sclerdium cepivorum. Subsequent effects of the cover crop residue incorporation on the
incidence of white rot and some other commonly occurring diseases in a succeeding crop
of commercial onions were also examined.
CHAPTER 2
EFFECT OF BRASSICA JUNCEA AS A WINTER COVER CROP ON THE SURVIVAL
OF SCLEROTIUM CEPNORUM
Crop rotation can affect the incidence of plant diseases (Curl. 1963; Griffin et d.
1981). Diseases caused by pathogens that decline rapidly in the absense of a host may be
controlled by enforcing an appropriate interval between successive occurrences of the host
crop. The same strategy can be used to slow the rate of population increase of an
introduced pathogen in the soil.
Some plant species can directly or indirectly affect the survival of pathogens of
other plants. Various researchers have found that the immediately preceeding crop in a
rotation, or certain organic amendments derived from crops, can have a substantial effect
on the incidence of diseases caused by different organisms (Butterfield et d. 1978;
Kollmorgen et d. 1983; Cook 1981c; Rarnirez-Villapudua and Munnecke 1988). Other C
studies have shown that during decomposition of crop residues in soil, volatile compounds ,
with phytotoxic as well as mycotoxic properties are produced (Lewis and Papavizas 1971;
Papavizas and Lumsden 1980; Patrick et d. 1964). Jarvis and Thorpe (1981) showed that
the use of lettuce as a cover crop between successive spring-sown greenhouse tomato
crops, and incorporation of the remaining lettuce residues into the soil controlled the foot
and root rot (crown rot) disease of tomato caused by Fusarium oxysgwum f. sp.
radicis lycopersici.
A modification of crop rotation for disease control is the use of a host species as
a 'trap crop' for a pathogen. The objective is to exploit the potential of the trap crop
to deplete the reservoir of resting propagules of the pathogen by stimulating their
germination, and then destroy the 'trap' or otherwise prevent reproduction of the pathogen.
Sclerdium cepivorum Berk., the fungus causing white rot of Allium spp., is
potentially an ideal candidate for the trap crop approach. Sclerotia of the pathogen are
able to survive in ' the field for many years in the absence of host Allium spp.
(Coley-Smith 1979; Crowe et al. 1980). so that conventional crop rotation would seem to
be of little value for reduction of inoculum. Sclerotia of S. cepivomm germinate in
response to volatile sulfide compounds derived from enzymatic or microbial action on
non-volatile precursors found uniquely in root exudates of Allium spp. (Coley-Smith &
King 1969; Coley-Smith & Cooke 1971). Merriman and Issaacs (1978) tested onions as a
trap crop for S. cepivorum Despite the apparent potential of the approach, they found
that onions did not significantly reduce populations of sclerotia in soil, even though
disease occurred in 19% of the trap crop plants and these were destroyed with a
herbicide prior to the appearance of new sclerotia
Coley-Smith and Parfitt in 1983 reported that germination of sclerotia of S.
cepivomm in non-sterile soil can be stimulated by allylisothiocyanate (ACN). This compound
does not occur in Allium spp., but is found in certain crucifers. Decomposing crucifers are A
known to produce substances with mycotoxic properties (Patrick 1986), and such substances
might also contribute to the death of germinated sclerotia. The purpose of this experiment
was to evaluate whether Brassica Jitncea (mustard), a crucifer, grown as a winter cover
crop might enhance the decay of sclerotia of S. cepivorum under field conditions. Brassica
Jitncea was chosen for study because it is known to produce ACN (Itoh et al. 1984.
1985; Kozima et al. 1987), and also for its ability to produce large amounts of leafy
biomass during the relatively mild fall and winter conditions that typically occur in the
Fraser Valley of British Columbia.
2.2 MATERIALS
The study
METHODS
site was in a field that is part of a commercial muckland vegetable
farm near Cloverdale, B.C. The field was cropped to onions and suffered losses of
25-30% due to white rot in 1986 (J.E. Rahe, personal communication). The field was
rototilled and rolled after harvest of the previous year crop. The study site was 270x36
m overall, and was divided into six plots of 45x36 m Seed of B. Jitncea, cv. Cutlass,
was kindly provided by Dr. G.F.W. Rokow, Agriculture Canada Research Station,
Saskatchewan. Alternate plots in the study site were seeded to B. Jitncea on 4 September,
1986 at 2.3 kg/plot using a Cyclone Little Giant hand seeder, and seeding in both
directions. Plants were disced down around 15 May, 1987.
Three random sites were selected in each of the B. Jitncea seeded plots on 22
February, 1987 for the purpose of estimating the amount of plant biomass. At each site
plants were pulled from a 929 cm2 area and carried to the laboratory in plastic bags.
All the soil was shaken off the plants, and the contents of each bag were weighed
separately. C
* After about two months storage of these mature plants in the refrigerator, volatiles
collected from rotting mature plant leaves were trapped on Porapak Q trap over a three
day period and analysed by gas liquid chromatography by H.D. Pierce, Jr, (Department of
Chemistry, Simon Fraser University).
Each plot was subdivided into 10 subplots for the purpose of estimating populations
of indigenous sclerotia. Composite soil samples representing each of the 60 subplots, each
consisting of a minimum of 35-40 g of soil collected from the surface and at
approximately 25-30 equidistant locations in a subplot, were taken on 4 September, 1986.
The study site was sampled in a similar fashion on 22 February, and 3 June, 1987 after
the Brassica plants had been disced down. Only 24 samples (Four subplots from each
treatment plot) were taken on 22 February, 1987. Each composite sample was mixed in a
rotary tumbler for 5 min at 48 revolutions/min. After mixing, a single 20 g (wet wt)
subsample was taken from each sample and the number of sclerotia contained was
determined by wet seiving and centrifugation in 70% sucrose (Vimard et al. 1386).
Moisture content of the soil was determined by drying 25 g of soil from each sample
(second and third sampling times) at l lo•‹C in an oven for 24 hours and reweighing.
Percent moisture was calculated as follows.
weight of wet soil - weight of dry soil
% moisture= x 100
weight of dry soil
Moisture contents of the soil samples at the first sampling time (September, 1986)
were not determined. The soil samples taken in September, 1986 were considered to have
the same moisture content as those obtained in June, 1987. Consequently the mean
moishue content of June samples was used for estimation of propagules on dry weight of
soil basis for the September, 86 samples.
Introduced sclerotia used in the study were obtained from naturally infected onions
from this farm. Sclerotia were harvested from bulbs and kept in soil in clay pots in the
laboratory at room temperature, and watered infrequently. Sclerotia were recovered from the
soil in pots by wet sieving when needed. Two populations of sclerotia were used, one
from onions produced in 1985 and aged in the laboratory for 1 year, and the other
from onions of the 1986 crop. Two hundred sixteen nylon mesh bags, each containing 20
intact firm sclerotia in 25 g of soil, were prepared. The soil was taken from a headland
of the field in which the study site was situated and was found by analysis to be free
of indigenous sclerotia. On the following day, one month after seeding, three bags from
each population were placed at each of six replicate sites in each plot. The bags were
buried 5-6 cm deep in the soil and attached to a central wooden stake at each site by
color coded nylon ribbons (Fig. 4).
Six replicate bags of each population (one from each stake) were recovered after 8.
19 and 25 weeks of burial. Sclerotia in each bag were recovered by wet sieving and
centrifugation, and their numbers and condition assessed. Intact, firm sclerotia were surface
sterilized in 1% NaOCl for 4 min, rinsed six times with sterile deionized water, plated
onto potato dextrose agar (PDA) and kept at 1 7 O C . The plates were observed for 15
days, and those sclerotia producing colonies typical of S. cepivotum were recorded as
viable.
Tests of viability for broken, geminating, soft or hollow sclerotia were not reliable
therefore for analysis of data only intact firm sclerotia were considered. The sclerotia
recovered from all samples taken for indigenous population estimation were recorded as
numbers/kg of dry soil. Data were analysed by analysis of variance and covariance (using
time as a covariate). Selected means were compared by using Student's T-Test (P=0.05).
FIGURE 4: Field experiment showing arrangement of nylon mesh bags containing sclerotia of Sclerotium cepivorum in a study plot
2.3 RESULTS
Excellent emergence of B. bncea occurred, and dense stands were present in the
seeded plots by October. These plants continued to grow during milder portions of the
fall and winter. By late February, above ground stems ranged from 39-65 cm in length,
and overall plant height from 56-84 cm. Typical stems were 2-3 an in diameter at the
crown. Fresh weight biomass (roots and shoots) averaged 438*60g/929 an2 (47 tomes/ha)
on February 22, 1987. Sparse growth of winter annuals. primarily chickweed (Stellaria
media) and some unidentified grasses occurred on the unseeded plots.
Distribution of sclerotia was patchy in each plot and variation among samples was
high in control and seeded subplots (Table 1). None of the sclerotia recovered were found
to be in a germinating stage. Temperature and rainfall data for the period September, 86
to June, 87 are shown in Table 2.
Mean populations of indigenous sclerotia at the time of seeding were 535 and 445
per kg dry soil in seeded and control plots, respectively, but the difference was not
statistically significant. The population of sclerotia decreased significantly between the first (4 P
September) and second (22 February) samplings in seeded but not in control. plots (Fig.
5, Table 1). Populations of sclerotia in both seeded and control plots appeared to decline
markedly between the second (22 February) and third (3 June) samplings. Much of the
apparent decline may have been due to dilution of the surface population of sclerotia
with subsurface soil caused by discing and rototilling that occurred about 15 May.
Estimated populations of indigenous sclerotia on 3 June, 1987 were 24.5 and 49.5 per kg
dry soil in seeded and control plots, respectively, and the difference between these was
not significant Less than one percent of recovered sclerotia were found to be soft and/or
germinating when sampled on 3 June.
The numbers of intact firm sclerotia recovered from bags containing known numbers
of sclerotia obtained from infected onions declined significantly with time in both seeded
and control plots (Fig. 6). Decline was slight until December, and accelerated during the
winter months. Most of the decline was due to missing or decomposed sclerotia. The
combined total of soft, broken and germinating sclerotia recovered at each sampling time
ranged from 0.8 to 2.9 percent of total numbers of intact sclerotia introduced. Survivorship
of sclerotia of the 1985 and 1986 populations buried in the test plots did not differ
(P=.4841). Numbers of introduced sclerotia in buried nylon mesh bags declined more
rapidly in the seeded plots than in the control plots. After 25 weeks, populations of
sclerotia buried in plots without B. Jitncea had declined by 38% while those buried in
plots with B. Jitncea had declined by 56% (Fig. 6) and were significantly different
(P<0.05). Viability of recovered sclerotia ranged from 91-100%. Most of the recovered
sclerotia that failed to produce colonies of S. cepivorum on PDA following immersion for
4 min in 1% NaOCl were contaminated with a bacterium that produced inhibition zones
against S. cepivwum when subsequently tested in dual cultures (Fig. 7).
Analysis of the volatiles released by decomposing plants of B. Jitncea harvested in C
February showed a number of constituents including a distinct peak of allylisothiocyanate ,
(H.D. Pierce, Jr, personal communication).
Table 1: Indigenous populations of sclerotia of Sclerotiurn cepivorurn estimated at 0, 171 and
272 days after seeding with Brassica Nncea.
Sclerotia recovered per kg dry weight of soil**
TREATMENT Sept 4, 1986 Feb. 22, 1987 June 3, 1987
0 days 171 days 272 days
BRASSICA 535f76.49 a 201f 55.36 b 24.829.78 c
(30, 0-1489.0) (12, 0-517.5) (30, 0-106.5) P
CONTROL 445f113.48 a 431f95.95 a 49.5f11.10 c
(30, 0-2872.5) (12, 0-1043.0) (30, 0-212.5)
Data are MEAN f SE, with n and range in parentheses.
*Means followed by same letter do not differ significantly as tested by student's T-Test
(P4.05).
Table 2: Temperature and rainfall data* for the period September, 86 to June, 87.
MONTH TEMPERATURE (O C )
mean range
RAINFALL
rnrn/month
Sep, 86
Oct
Nov
Dec
Jan, 87
Feb
Mar
A P ~
May
Jun
* Data obtained from Environment Canada, Surrey Municipal Hall. B.C.
Figwe 5: Effects of Brassica bncea, natural conditions and rototilling on persistence of
indigenous populations of sclerotia of Sclerotium cepivorum in Fraser Valley muck
soil.
Legend eZd BRASSICA PLOTS
CONTROL PLOTS
S O N D J F M A M J 1986 1987
TIME OF THE YEAR
Figure 6: Effects of Brassica juncea and natural conditions on the survival of introduced
sclerotia of Sclerotium cepivorum in Fraser Valley muck soil.
Legend 0 1985 in Brassica ---- a 1986 in Brassica -------------------
1985 in Control ---- 1986 in Control
OCT NOV DEC JAN FEB MAR APR 19 8 6 1987
TIME
Figure 7: Dual culture test on potato dextrose agar showing antagonism between Sclerotium cepivorum and an unidentified bacterium isolated from the surface of sclerotia of
S. cepivorum.
2.4 DISCUSSION
The results reported here show that sclerotia of S. cepivorum decayed substantially
during a single winter season in the Fraser Valley mucklands. This supports the findings
of Leggett et al. (1983)' and Leggett and Rahe (1985) but are in contrast to the
observations of Coley-Smith (1979) and Crowe et al. (1980). Leggett (1983) suggested that
the high rate of decay of sclerotia in the Fraser Valley might be associated with winter
flooding typically encountered in Fraser Valley muck soils. However, she presented only
indirect evidence for this contention.
The results of the present study showed that sclerotia decayed significantly with time
in all plots and more rapidly in plots seeded with B. Jitncea (Fig. 6). This effect was
observed for the indigenous sclerotial population and for two populations of introduced
sclerotia. This suggests that the presence of the B. juncea crop influenced survival of
sclerotia of S. cepivorum.
The addition of organic matter or crop residues has been used to control many
diseases (Huber et al. 1970). Lewis and Papavizas (1977) studied the effect of decomposing
immature and mature residues of rye, oat, soybean, sorghum, barley, buckwheat, timothy
and corn on Fusarium root rot of bean and found that most of these residues
significantly reduced the disease. Toussan, Patrick and Snyder (1963) studied the effect of
crop residue decompostion products on the germination of Fusarium sdani f. sp. phasedi
chlarnydospores in soil and found that these reduced the inoculum levels by inhibiting
chlamydospore formation. Many studies have shown that during decomposition of crucifers
in the soil, compounds with phytotoxic and/or mycotoxic properties are produced (Patrick,
1986). Some of these substances had strong inhibiting effects on mycelial growth. zoospore
motility and germination of infective propagules of Aphanomyces euteiches and reduced pea
root rot (Lewis and Papavizas 1971).
The results of this study would suggest that B. /itncea released certain compounds
which stimulated sclerotial germination of S. cepivorum in the absence of host Allium spp.
Coley-Smith and Parfitt (1983) studied the response of sclerotia of S. cepivorum to
artificial stimulants and found that allylisothiocyanate applied to soil stimulated up to 66%
germination of sclerotia compared to 3% germination in the control. Allylisothicyanate and
diallyldisulphide were comparable in their ability to promote germination of sclerotia of S.
cepivorum. Surprisingly, allylisothiocyanate is not a component of Allium spp. B. /itncea has
been found to contain the highest levels of allylisothiocyanate among all the crucifers
studied so far, and its presence was confirmed in the vapors emanating from rotting
plants produced in this study (H.D. Pierce, Jr, Personal communication). Accordingly it
seems possible that the presence of the B. Jitncea crop in the field might have induced
or stimulated germination of sclerotia, perhaps because of the presence or release of
allylisothiocyanate and the germinated sclerotia decayed rapidly in the absence of a suitable
host
The recovery of intact firm sclerotia decreased significantly with time (Fig. 5 and
Fig. 6 ) in all cases. It is not known yet if this decay or disappearance of sclerotia was C
due to natural flooding encountered in the field (Leggett & Rahe 1985), the presence of
organic matter, the effect of which could be general because of increased microbial activity
resulting in increased antagonism, or specific due to certain volatiles from B. &ncea that
stimulated the germination of sclerotia as well there is the possibility that decomposition
of organic matter or crop residue from B. Jitncea might have accelerated oxygen
consumption by soil microflora in the saturated muck soil. In such soil sclerotia might
have died as a consequence of oxygen depletion. A detailed study on the ef'fect of
flooding and oxygen requirements for survival of sclerotia under natural environmental
conditions is needed to evaluate the above hypothesis.
CHAPTER 3
SURVEY OF DISEASE LEVEL IN COMMERCIAL ONION FIELD
3.1 INTRODUCTION
The ultimate aim of efforts made to reduce inoculum levels of soil-borne pathogens
is to reduce the diseases caused by those pathogens to economically acceptable levels. It is
difficult to eradicate the inoculum completely, particularly if it is soil associated. Papavizas
& Lumsden (1980) noted out that if inoculum levels are reduced, the efficacy of other
control methods (cultural practices, resistant or tolerant cultivars, fungicides) may be
enhanced. However, it is, difficult to state what is an acceptable level of inoculum
because it differs among different host species, pathogens, soil and environmental conditions.
The study described in the previous chapter demonstrated that B. /itncea grown as a
winter cover crop in 1986-87 reduced the survival of sclerotia of Sclerdium cepivorum
significantly compared to controls. So next step was to see if this reduction in population
of sclerotia in treated plots, reduce the level of disease in these plots @ in the succeeding
onion crop. This chapter describes an analysis of the survey done on the estimation of
the diseases like white rot and smut and the yield of healthy marketable onions in a
crop of commercial onions grown on the previous study site in 1987.
3.2 METHODS FOR ESTIMATING DISEASE LEVEL
The experimental site described in the previous chapter (Brassica trial) was seeded
with the dry bulb onion cultivar "Aries" on 28 May, 1987. The onions were planted in
five rows on raised beds with a Stanhay precision seeder. There were nine beds running
the length of the study site and crossing each of the six subplots. Indigenous sclerotial
population in the study site was assessed 19 days after incorporating the cover crop
residue as described in chapter 2.
Six of the nine beds of the onion crop were assessed for white rot, smut and
other diseases at various intervals beginning 5 August, 1987. A systematic pattern of
sampling was adopted for this survey by taking four, approximately equidistant samples
from each bed in each subplot. All sampling sites were located at least 3 m from the
boundries separating the subplots. At each sampling site a plant in a row was chosen at
random, and 10 contiguous plants were taken and rated for smut, white rot and other
diseases such as maggot and other unidentified diseases of onions. Samples were taken on
5, 12, 20, and 26 August, and 4 September, 1987. For analysis of white rot data, sample
sites were scored as uninfected, or infected, if any plants with white rot were found
among the 10 contiguous plants. In the case of smut, the number of plants with smut
was recorded. Data were subjected to analysis of variance with nested design using time
as a covariate. Selected means were compared by using a student's T-test (P4.05).
On 21 and 22 September, 1987, a more extensive sampling was done. At each of
five sampling sites in each of the six beds in each subplot, 0.25 m2 quadrats were
assessed for total number of plants present, the number of plants wlth white rot, smut P
and other unidentified diseases including maggot, and the yield of healthy. marketable '
bulbs. Data were analysed by analysis of variance, and means were separated by using
DMRT.
Immediately after the onions were harvested, on 17 October, 1987, composite soil
samples were taken for estimating populations of indigenous sclerotia from the experimental
site and from an adjacent area that had been cropped to potatoes in 1987. Twenty
composite soil samples were taken from each field (40 total). Each sample consisted of
20-25g of soil taken 0-6 cm deep from 25-30 approximately equidistant locations in each
plot. Each composite sample was mixed in a rotary tumbler for 5 rnin at 48
revolutions/min. After mixing, 25g and 20g (wet weight) subsamples were taken from each
sample, the former for estimating moisture content and the latter for estimating sclerotial
populations. The numbers of sclerotia contained in the soil samples were determined by
wet sieving and centrifugation in 70% sucrose (Virnard et al. 1986). Moisture content was
estimated as described in chapter 2. Recovered sclerotia were assessed for condition, and
intact firm sclerotia were' surface sterilised with 1% NaOCl for 4 rnin, and plated onto
potato dextrose agar. Their viability and identity was assessed after incubation at 17-18 '~
for 2 weeks. Numbers of sclerotia were calculated on dry wt basis and converted to
nurnbers/kg dry soil, and tested for difference in populations of sclerotia between the
onion and potato fields by using a student's T test (P=0.05)..
3.3 RESULTS
Preliminary sampling by Dr. J.E. Rahe on 20 July 87 (personal communication), and
the samples taken on August 5, 1987 detected very few plants with white rot (maximum
four plants in 1440 plants sampled), but smut was widespread. The level of white rot
increased dramatically in samples taken after 5 August Analysis of data from samples
taken August 12 to September 4, 1987 (4 sampling times) showed that the level of white
rot did not differ significantly with time between August 12 and September 4 (Table 3,
T-test). Contrary to expectations based on the significantly enhanced decline of introduced
sclerotia observed in Brassica subplots compared to control subplots (Fig. 6, Chapter 2).
analysis of the pooled data for the period August 12 to September 4 showed that
significantly higher levels of white rot occurred in treatment subplots than in control
subplots (Table 4, T-test).
Smut levels appeared to decline with time after the first sampling (Table 5),
presumably due to drying and loss of the small plants affected by smut Percent smut
was numerically less in treatment than in control plots on four of the five sampling C
dates, but the difference was significant (T-test) only for the 12 August sampling. ThereP
was a significant block effect for both white rot and smut The gradient of smut and
white rot in blocks was found opposite to each other.
Harvest data taken on 21 and 22 September, 1987 did not show a significant
treatment or block effect for white rot, nor a significant treatment effect for smut (Table
6, T-test). Other pests including maggot and some unidentified onion diseases were detected
on 4.55 percent of the total plants present and were not significantly affected by
treatments but did show significant differences among blocks. Yield was significantly
different in the three blocks, but the treatment and control plots did not show significant
differences (P=0.05). On average the yield of healthy marketable onions from treated and
control plots was 31.58 and 28.85 tomes per hectare, respectively (Table 6).
Mean populations of indigenous sclerotia when sampled on 17 October were 37.3 and
39.8 per kg dry weight of soil in the potato and onion (treated and control) fields,
respectively, and the difference between these was not significant. Numbers of sclerotia
recovered in individual samples ranged 0-3/20 g wet weight of soil for both fields.
Table 3: Comparison of proportions of sites* with white rot at different sampling times in
Brassica treated and control subplots.
TREATMENT Aug.5 Aug.12 Aug.20 Aug.26 Sept4 Sept22
BRASSICA
CONTROL
*The values are means of three blocks and six beds.
Table 4: Comparison of percentage* of plants with white rot at different sampling times in
Brassica treated and control plots.
TREATMENT AugJ Aug.,l2 Aug.,20 Aug.,26 Sept,4
BRASSICA 0.00 5.00 10.69 9.44 3.33
CONTROL 0.42 3.33 6.38 $25 2.63
L
Values are means of three blocks and six beds.
Table 5: Comparison of percent smut* at different times in Brassica treated and control plots.
TREATMENT
BRASSICA
CONTROL
fl
Values are means of three blocks and six beds. L
* Means were significantly different (T-test, P=0.05) only for 12 August sampling.
Table 6: Comparison of disease levels and yield of onions at harvest* in Brassica treated and
control plots.
TREATMENTS Percent Percent Percent Yield in
White rot Smut Other Tomes Per
Diseases Hectare
BRASSICA
CONTROL 12.95 9.38 4.61 28.85
* Values are means of three blocks and six beds.
3.4 DlSCUSSION
The results obtained during this survey show that the level of white rot was higher
in the treated (Brassica) plots than in control plots from 12 August to 4 September. This
difference apparently disappeared late in the season, as the harvest data obtained on 21
and 22 September, 1987 showed no significant differences among treated and control plots
on white rot incidence (Table 6).
The data show that visible signs of white rot on onion bulbs developed within a
two weeks period (Aug., 5 to Aug., 20). The sudden, late. season appearance of white rot
has generally been observed in the Fraser Valley, as has also the general observation that
if the crop is sown early, white rot shows up early and if it is sown late, white rot
shows up late (Dr. J.E. Rahe, personal communication). Moreover, on this particular farm,
a field adjacent to the study site that was seeded in late March of 1987 developed
white rot in early June (M. Tardif & J.E. Rahe, unpublished observation). These
observations suggest that infections resulting in visible signs of disease may occur at a
particular stage of host development or that latent infections may be present in an C
apparently healthy crop, and that development of visible mycelium on the bulb may awaitP
certain physiological changes of the host
A possible explanation for the increased incidence of white rot in the treated plots
during the early stages of disease development is that the Brassica residue may have
served to activate propagules of the pathogen. The 1987 onion crop was seeded within
two weeks of incorporating the B. juncea crop residue into the soil. Its decomposition
could have stimulated germination of sclerotia via release of allylisothiocyanate (ACN,
Coley-Smith and Parfifitt 1983). Without a source of ACN in the control plots, sclerotial
germination there would depend on production of volatile stimulants by the host, and
hence occur later and possibly at lower levels than in the treated plots.
The effect of treatment (B. hncea cover crop and its subsequent incorporation into
the soil) in reducing the incidence of smut uncovers some other unknown, possible
influences of B. hncea on other diseases of onions. It is hard to say at this stage if
there is any negative correlation between smut and white rot incidence. As blocks showed
significant differences, it 'shows that the experimental field was not homogenous, which
could be due to many environmental factors.
The means of populations of sclerotia did not show significant differences between
field cropped to onion and potato, which shows that the subsequent host crop (onion) did
not influence the inoculurn levels of S. cepivorum in 1987. This unexpected result is likely
related to the restricted development of disease in 1987. Overall, white rot infections in
1987 were found to be on 12.3% of the plants sampled whereas it was 25-3096 in 1986
(J.E. Rahe, personal communication). The major difference between disease development in
1986 and 1987, however, was that in 1986 most of the bulbs that were infected with S.
cepivorum had black sclerotia formed already by early August, whereas in 1987 even at
the time of harvest (Sept, 21, 22) there were very few bulbs (approximately 1%2% of
infected bulbs) that had black sclerotia formed on them. It appears that very few sclerotia @
were formed as a result of the white rot that developed in 1987. This could be due to*
some environmental conditions (high temperature and hence low moisture) that occurred in
1987.
B. hncea as a winter cover crop, did not reduce white rot or improve the
subsequent onion crop despite its effect on the rate of decline of sclerotia of S.
cepivorum, neither did it have an adverse effect, as the yields obtained from treated and
control plots did not differ significantly. If the hypothesis that the B. /itncea residue
served to enhance germination of sclerotia and hence infection of onion seedlings is
correct, it is possible that reduced disease might result if the Brassica were incorporated
in early winter of the preceding year, or if seeding of onions were delayed for one
year.
CHAPTER 4
EFFECT OF FLOODING AND ORGANIC AMENDMENTS ON SURVIVAL OF
SCLEROTIA OF SCLEROTIUM CEPIVORUM
INTRODUCTION
Changes in the physical and chemical environment of the soil can directly or
indirectly affect the infection and development of disease in plants. They may directly
influence plant health, or affect the survival and/or infection abilities of pathogens, hence
influencing disease incidence. Properties of the soil environment include soil texture, organic
and biological composition, pH, temperature, aeration and gas exchange, water and nutrient
availability, and other chemical factors; these can be effected by crop rotation, tillage
practices, and crop residue management
Soil water can influence the incidence of disease both directly and indirectly. It is
one of the most poorly understood aspects of the soil environment as regards its
influence on microbial interactions in soil. Infection and development of disease in plants C
can be altered by changes in soil resulting from waterlogging. Cook and Papendick (1972).
stated that fungi are favoured by a relatively dry environment and bacteria by wet
conditions. Excessive soil water is often harmful to higher fungi and waterlogging could
eliminate some sensitive species (Mengenot & Dien 1979). The lower fungi are an
exception and require free water for zoospore movement Stover (1979) reported that only
one species of bacteria, Pseudomonas sdanaceamrn, on tobacco (n7icotiana) and bananas
(Uusa) was controlled by flooding. Several diseases caused by fungi have been controlled
by maintaining a flooded soil for varying periods of time. The Chinese have long
recognized the benefits of rotations of paddy rice with cotton for control of Fusarium
oxysporum f. sp. vasinfectum (Cook, 1981b). Moore (1949) found that sclerotia of
Sclerotinia sclerotiorurn decayed completely within 23-45 days when marl, muck or sandy
soils were flooded. Another classic example of control of disease by flooding is that of
banana wilt, caused by Fusarium oxyspwurn f. sp. cubense in Central America (Stover
1953).
The rate at which a soil becomes anaerobic when saturated with water (flooding) is
largely related to the rate of oxygen utilization which in turn is related to the level of
aerobic microbial activity. Microbial activity is generally enhanced by addition of organic
amendments. The availability of nutrients or organic matter in a soil is often related to
pathogen suppression (Menzies 1962 & 1970; Moore 1949; Smith & Restall 1971 and
Watson 1964). For example, when microsclerotia of Verticillium dahliae were incubated in
saturated soil under nitrogen, the microsclerotia were eliminated in 3 weeks. When 1%
alfalfa meal or 0.1% glucose was added, the microsclerotia were eliminated in 5 days
(Menzies 1962). These results suggest that addition of organic matter or a food supplement
might enhance decay of a pathogen, caused by flooding.
Fraser Valley muck soils are often flooded during the winter season (Leggett &
Rahe, 1985), and the unusually high rate of decay of sclerotia in these soils was
attributed to winter flooding (Leggett et al. 1983). Valdes and Edcgington (1987) also P
observed reduced incidence of white rot in flooded soil. The purpose of this experiment
was to study the effect of flooding and organic matter incorporation on the survival of
sclerotia under conditions typical of the winter months in Fraser Valley. Earlier studies
showed that recovery of sclerotia from plots seeded with Brassica jimcea was less than
from unseeded plots (Fig. 6), B. &ncea and indigenous winter annuals were compared as
organic amendments.
4.2 MATERIALS METHODS
This study was conducted outdoors at Simon Fraser University, Bumaby, B.C.
Styrofoam cups of 350 rnl capacity were used as experimental pots. The bottoms were cut
open to allow drainage iri half of the cups and half of the cups were kept intact for
the flooding treatment (Fig. 8).
Nylon mesh bags were prepared, each containing 20 intact firm sclerotia with 20 g
of unsterilised, sclerotia free muck soil. The sclerotia were of the 1986 population
described in chapter 2.
Brassica plants brought from the experimental field site described in chapter two
were chopped into about 5 cm pieces and added to each cup at a rate equivalent to
the amount of biomass present in the field, which turned out to be 20 g fresh wt per
cup. Indigenous winter vegetation from the control plots (grass and chickweed) was
prepared in a similar manner and added to other pots, also at 20 g/cup. Into each cup
some unsterilised muck soil was placed, followed by half of the plant material (where
added), the nylon bag containing sclerotia, the remaining plant material qwhere added), and *
finally &e cup was filled with soil to approximately 3/4 of its volume (Fig. 9). Pots for
nonflooded treatment were prepared on site.
There were total of 6 treatments (3 flooded and 3 nonflooded) and 8 replications
of each treatment and the pots were arranged in a Randomized Complete Block design
(Fig. 10) outdoors under natural environmental conditions. The experiment was set up on
February 23, 1987. At the beginning of the experiment, the viability of sclerotia from the
population used for the experiment was estimated on potato dextrose agar by the method
described in chapter 2.
Figure 8: Styrofoam cups to show the cut and intact bottom used for drained and flooded condition, respectively.
Figwe 9: Diagramatic representation of arrangement of nylon mesh bags containing sclerotia of Sclerotium cepivorum and plant material in the soil in cups.
NYLON BAG t
WITH SCLEROTIA
BRASSICA OR VEGETATIVE MATERIAL
UNSTERILISED SOIL
After 90 days, the bags were removed from the cups and the contents were
analysed for percent recovery and viability of sclerotia. All bags were analysed within a 4
day period. Only intact firm sclerotia were considered for data analysis. Data were
subjected to analysis of variance and means were separated using Duncan's Multiple Range
test
Figure 10: Experimental setup showing flooded & nonflooded treatments in a randomized complete block design.
4.3 RESULTS
The results of this study are presented in Fig. 11. The percent recovery of intact
firm sclerotia decreased significantly (Pc0.05) within 90 days in all treatments except
unflooded unamended and ' unflooded but amended with indigenous vegetation. The results
showed significant effect of flooding and Brassica or vegetation incorporation in reducing
survival of sclerotia over controls.
This study showed that flooding favours decay or disappearance of sclerotia (Fig. 11).
It also suggested that decay of sclerotia was enhanced when some organic matter was
added to the flooded pots, in comparison to the nonamended treatment The results
indicate that organic matter from B. juncea added in flooded soil significantly decreased
the percent recovery of sclerotia In this treatment the population of sclerotia was reduced
to 10% of its original population, where as in nonflooded replication of this treatment, it
declined to 64.8%.
There was a clear difference in sclerotial recovery between Brussica treated and
general vegetation treated pots. Many sclerotia recovered from flooded fieatments lost their L
integrity and were very soft. Some plug germinating sclerotia were also recovered. Viability
of sclerotia recovered from all six treatments ranged from 87.5% to 97.8%. Viability of
sclerotia at zero time was 100%.
As was found in the field study, most of the recovered sclerotia that did not
germinate on potato dextrose agar were contaminated by a bacterium that appeared to be
one of the type which survived surface sterlization with NaOCl solution and produced
inhibition zones against sclerotia when tested in dual cultures PDA.
Figure 11: Effects of organic amendments on survival of introduced populations of sclerotia of S. cepivorum under the influence of flooded or nonflooded soil.
Legend C S l BRASSICA PZa GRASS EW CONTROL
4.4 DISCUSSION
The use of flooding to control plant diseases was attempted in the muck soils of
Southern Florida where the root knot nematode is a problem in vegetable production
(Kincaid 1946). Although flooding has been known to cause a rapid decline in the fungal
population of the soil (Stover et al. 1953), there are only three documented cases of it
being used for fungus disease control. The diseases are: tobacco black shank in Sumatra,
Sclerotinia on celery in Florida and Fusarium wilt of bananas in Central America (Stover
1979; Stover et al. 1953).
Sewell (1965) pointed out that the responses of soil fungi to changes in soil
moisture are seldom attributable solely to the direct effect of water on the fungus of
concern. He stated that inadequate gas exchange, resulting in depletion of 0, and increase
in CO, concentration, probably was the primary cause of the inhibition observed in
saturated and very wet soils, both low O2 and high CO, concentrations have been shown
to inhibit the production of microsclerotia by Verticillium dahliae (Ioannou et al. 1977).
The Chinese have practiced rotation of paddy rice with cotton for control of Fusarium C
oxysporum f. sp. vasiinfectum (Cook 1981b). European grape growers have also used A
flooding to control the Phylloxera root aphid (Newhall 1955).
The results of this study provide direct evidence that flooding enhances decay of
sclerotia of S. cepivorum, and supports the hypothesis of Leggett and Rahe (1985) that
flooding is the factor responsible for high rate of decay of sclerotia in local muck soils.
Watson (1964) found that addition of several crop residues to Pyrenochaeta terrestris
infested soils resulted in reduction of the incidence of onion pink root He pointed out
that a combination of a proper residue and anaerobic fermentation was required for
eradication. He reported that sphagnum moss and dried soybean were not as effective as
green soybean, sweet clover, corn, timothy, alfalfa, oat, straw and sugar beet residues in
eradicating P. terrestris. Results from my study also indicate that the presence and nature
of organic residue interacts with flooding. Decay of sclerotia of S. cepivwum was higher
in soil amended with Brassica Jirncea residue compared with general vegetation (Fig. 11).
Flooding alone did not reduce the survival of sclerotia to the extent that it did when
the soil was amended with organic residue. Valdes and Edgington (1987) studied the effect
of crop rotation and flooding on the incidence of white rot and found a significant
decrease in flooded plots. They reported that when flooding was followed by rotation with
m o t s , comparatively better control of white rot was obtained in organic soil than by
flooding alone.
The results of the studies conducted here demonstrated that the population of
sclerotia was reduced to almost 10% of the original population in pots that were flooded
with Brassica amendments as compared with flooded pots with grass amendment where the
population was reduced to about 32%. It wouls seem that B. Jirncea might have some
stimulatory effect on germination of sclerotia, and that its effect is more pronounced when
the soil is flooded. It is possible that a combination of these two treatments could
provide a good control of white rot in the Fraser Valley muck soil. However, before this @
technique can be applied in disease management in the field, a detailed study needs to
be done in the field by flooding the field followed by crop rotation with B. Jitncea
There is possibility that flooding might have enhanced anaerobic decomposition of B.
/itncea which released allylisothiocyanate and stimulated sclerotial germination in the soil in
the absence of the host plant. Another aspect to be considered in this soil experiment is
the possible effect of antagonistic or competitive microflora. As has been found in this
study a bacterial isolate dominated as sclerotial surface microflora and was antagonistic to
S. cepivorum in dual culture test; it might be due to increase in this particular bacterium
that resulted in enhanced decay of sclerotia.
The decline in recovery of sclerotia over 90 days of intermittent flooding observed
in this study could be due to anaerobic conditions due to a depletion of oxygen and/or
increase in carbon dioxide, increased antagonism, competition, release of a specific
germination stimulant or a combination of all these factors. There seems to be a potential
for using B. @ncea as a' rotation crop combined with flooding the field for the control
of S. cepivorum on onion. As temperature is another important factor influencing the
effect of flooding in reducing plant pathogens, a study dealing this relationship needs to
be done.
CHAPTER 5
GENERAL DISCUSSION AND CONCLUSIONS
Biological and cultural control techniques have been used effectively to control certain
plant diseases (Huang 1980; Papavizas and Lumsden 1980; Curl 1963; Katan 1981; Sewell
1965; and Stover 1979). This area of plant disease control is receiving increasing attention
relative to chemical control. Chemical controls are generally expensive, can pose health
hazards in handling and product consumption, and may become ineffective after repetitive
use if the pathogens develop resistance to them. Reliable alternative methods of disease
control are being sought
Biological and cultural methods have provided effective control of white rot of
onions in some cases (Merriman and Borkenhead 1977; Smith 1972c; Utkhede & Rahe
1980) but results have been disappointing in other cases (Memman et al. 1980; Utkhede
& Rahe 1982) Germination of sclerotia of Sclerotium cepivorum, has been found to be
stimulated in field soil by host root exudates (Coley-Smith & King, 1969). Crop rotation
with non-host crops does not seem to have much potential for reducing the disease @
severity because sclerotia of S. cepivorum can survive for many years in the absence of .-
the host (Coley-Smith 1979; Crowe et al. 1980) Attempts have been made to use onions
as a trap crop for control of white rot (Merriman & Issacs 1978) but the results were
not very encouraging. However, the persistent population of sclerotia of S. cepivorum
remains an obvious target for biological/cultural control.
This study evaluated ways to reduce the populations of sclerotia of S. cepivorum in
naturally infested soil representative of commercial onion producing areas of the Fraser
Valley of B.C. using different agronomic practices. An analysis of the effects of Brassica
Nncea grown as a winter cover crop on survival of sclerotia of S. cepivorum showed
that it reduced the survival of sclerotia significantly. The presence of B. Nncea might
have stimulated germination of sclerotia which in the absence of a host decayed within a
short period of time. Obviously parasitized sclerotia were not recovered and viability of the
recovered sclerotia throughout the experiments was greater than 90%. Thus the treatments
did not reduce the viability of the remaining sclerotia. They presumably induced
germination of sclerotia Which, in the absence of a host, decayed rapidly due to lysis or
increased activity of antagonists or by some other mechanism. Direct evidence concerning
the mechanism of population reduction is lacking, however. and further studies should be
conducted to get a better understanding of the specific action of B. &ncea on S.
cepivorum.
The results from the survey of the indigenous sclerotial population raise certain
questions. They showed that there was a marked reduction of sclerotia of S. cepivwum in
treated plots between the first and second samplings (Table 1). There was even greater
reduction in both treated and control plots between the second and third sampling, such
that populations in the treated and control plots did not differ significantly when last
sampled in June. The study site was rototilled just before the last soil samples were
taken. It is possible that the unexpected precipitous decline in numbers @ of sclerotia in
both treated and control plots between the February and June samplings was due to .-
dilution of surface sclerotia with sub-surface soil as a result of rototilling. If this is true,
then it suggests that most of the sclerotia of S. cepivorum situated 210 crn below the
soil surface decay during the winter in our local muck soils. Furthur studies should be
directed to this hypothesis, and the effect of rototilling the land to different depths on
survival of sclerotia could also be evaluated.
Leggett and Rahe (1985) suggested that the high rate of decay of sclerotia of S.
cepivorum in Fraser Valley muck soils was presumably due to winter flooding. My study
suggests that the sclerotia decayed under the influence of flooding as well as crop residue
incoporation from the cover crop. Watson (1964) reported that the addition of several crop
residues to soil infested with Pyrenochaeta terrestris resulted in reduction of the incidence
of onion pink root Valdes and Edgington (1987) also reported that crop rotation of
onions with carrots reduced incidence of white rot in their muck soils.
The results of a survey done on the level of white rot and other diseases
occurring in the onion crop seeded immediately after incorporation of the B. Jirncea cover
crop suggested that despite the significant reductions of introduced populations of sclerotia
of S. cepivorum, white rot incidence was not significantly different in treatment and
control plots. The results also showed an unexpected effect of B. pncea in reducing smut
on onions. Yields of healthy marketable onions did not differ significantly in treated and
control plots.
Populations of sclerotia in the onion field and an immediately adjacent potato field
did not differ significantly after harvest of these crops, suggesting that the onion crop did
not increase the inoculum level in 1987. An unusual observation in the 1987 onion crop
was that the bulbs affected with white rot did not have black sclerotia formed on them.
Late planting and/or comparatively high summer temperatures in 1987 might have been
responsible for this. It is possible that as the fungal mycelium on b&s developed late in
the season, the mycelium might not have had time or experienced the conditions necessaryA
to form sclerotia. It has been generally observed that if the onions are seeded early,
white rot shows up early, and if they are seeded late, white rot shows up late in the
Fraser Valley (Dr. J.E. Rahe, personal communication). A detailed strldy dealing with the
optimum temperature conditions for white rot development in Fraser Valley muck soils
needs to be done.
The effect of intermittent flooding on survival of sclerotia in non-amended and soil
amended with B. Jirncea or natural vegetation was assessed. Flooding reduced the survival
of sclerotia but the magnitude of this effect was related to the presence and nature of
the vegetative amendments. This study did not provide direct evidence regarding the effect
of flooding and B. Jitncea residue incorporation on white rot incidence. Furthur studies on
this direction might help to improve our understanding of the interaction of undecomposed
organic matter and flooding on white rot in local muck soils.
My results agree with the findings of Valdes and Edgington (1987) who reported
that a combination of flooding with crop rotation provided better control of white rot
compared to flooding alone. Cook and Papendick (1972) stated that wet soil favours the
development of many diseases but completely saturated soil can have a detrimental effect
on the survival of many pathogens. Sclerotia of Sclerotinia sclerotiorum has been reported
to lose their viability within 60 days in saturated muck soil (Brooks 1939). Flooding of
banana land to eradicate Fusarium oxysporum f. cubense in Central America is another
example of good disease control (Stover 1979).
My studies show that flooding and incorporation of a specific residue can be used
to control the population of sclerotia of S. cepivomm in the soil. Garrett (1944) pointed
out that adequate control of some pathogens could be accomplished by reduction of the
numbers of the pathogen to a low level rather than its complete elimination. The results
from this study have clearly shown that flooding enhances sclerotial decay and the effect A
is significantly greater when the soil is amended with specific crop residues. If the
hypothesis that B. hncea residue served to enhance germination of sclerotia and hence
infection of onion seedling is correct, it is possible that reduced disease might result if
the Brassica were incorporated in early winter of the preceding year, or if seeding of
onions were delayed for one year. Unfortunately I could not follow up with this
evaluation as the farmer who provided the experimental plot decided not to grow onions
on the study site in 1988.
A considerable reduction of population of sclerotia of S. cepivorum could possibly be
achieved in Fraser Valley. muck soils by growing B. Jitncea as a winter cover crop,
incorporating the crop residue in the soil and then flooding the field. Temperature is also
an important factor influencing the interaction of residue amendments and flooding and a
detailed study on the effect of flooding and Brassica residue incorporation at different
temperatures is needed.
Flooding, particularly during the winter months could easily be implemented in the
Fraser Valley. B. /itncea grows well under the local conditions, produces heavy biomass
and is an economically feasible crop to be used as a winter cover crop. For practical
application of this approach, consideration must be given to the facts that B. /itncea is
highly susceptible to club root (Plasmodiophora brassicae), and is a very good host for
turnip mosaic virus.
In summary, I have shown that flooding enhances decay of sclerotia of S.
cepivorum. When specific organic materials are incorporated into the soil which is flooded,
a significant reduction in sclerotial populations can be achieved. This effect might be used
to reduce yield losses to white rot of onions under our local environmental conditions,
although I did not demonstrate this potential in my study. Results from my study are
largely preliminary and represent the first initiative taken towards the possible effect of
flooding and B. Jitncea on survival of sclerotia of S. cepivorm in thc Fraser Valley muck P
soils.
These findings open some new areas for furthur studies. More work is needed to
fully understand the mechanism of action of flooding and B. /itncea on survival of
sclerotia of S. cepivorum, and the interaction of these factors with temperature. Subsequent
research in these areas may lead to a better and more reliable approach to white rot
control than presently available.
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INDEX
ABSTRACT, iii ACKNOWLEDGEMENTS, v BIBLIOGRAPHY, 58 BIOLOGICAL CONTROL, 10 CHEMICAL CONTROL, 4 DEDICATION, vi DISCUSSION, 27, 38, 50 DISEASE HISTORY AND INTRODUCTION TO THE THESIS, 1 EFFECT OF BRASSICA JUNCEA AS A WINTER COVER CROP ON THE SURVIVAL
OF SCLEROTIUM CEPIVORUM, 14 EFFECT OF FLOODING AND ORGANIC AMENDMENTS ON SURVIVAL OF
SCLEROTIA OF SCLEROTIUM CEPIVORUM, 41 FACTORS AFFECTING GERMINATION OF SCLEROTIA, 6 GENERAL DISCUSSION AND CONCLUSIONS, 53 INFECTION AND DISEASE DEVELOPMENT, 3 INTRODUCTION, 14, 29, 41 MATERIALS AND METHODS, 16, 43 METHODS FOR ESTIMATING DISEASE LEVEL, 29 PATHOGEN. 2 RESULTS, 20, 32, 48 SURVEY OF DISEASE LEVEL IN COMMERCIAL ONION FIELD, 29 SURVIVAL OF THE PATHOGEN, 2 SYMPTOMS OF THE DISEASE, 4 WHITE ROT, 1
